Small organic molecules of interest to the biotech industry often involve aromatic structures that are derived from p-coumaric acid (pHCA) or other hydroxycinnamic acids. In particular, pHCA is a precursor for many secondary metabolites including flavonoids and stilbenes, and has a significant potential as a building block for producing polymers. pHCA is naturally formed from phenylalanine by subsequent ammonialyase and hydroxylase reactions or directly from tyrosine by the deamination of tyrosine.
Aromatic amino acid lyases constitute an enzymatic family, and are classified by their substrate specificity as being histidine ammonia-lyases (HAL, EC 4.3.1.3), tyrosine ammonia-lyases (TAL, EC 4.3.1.23), phenylalanine ammonia-lyases (PAL, EC 4.3.1.24) or phenylalanine/tyrosine ammonia-lyases (PAL/TAL, EC 4.3.1.25). Enzymes categorized as acting on either of the structurally similar amino acids tyrosine or phenylalanine are normally having some activity towards the other (Rosier et al., 1997; Zhu et al., 2013). Similar enzymatic families are tyrosine 2,3-aminomutases (TAM, EC 5.4.3.6) and phenylalanine aminomutase (PAM, EC 5.4.3.11) (Christenson et al., 2003a; Jin et al., 2006). All of these proteins contain a prosthetic group, 3,5-dihydro-5-methylidene-4H-imidazol-4-one (MIO) formed by the cyclization of the sequential three amino acids alanine, serine and glycine. TAMs as well as PAMs have been shown to have background lyase activity (Christenson et al., 2003b; Walker et al., 2004). The lyase and mutase activities of a single enzyme may be subject to a thermal switch (Chesters et al., 2012), and mutations can divert the enzymatic activity of a PAM into higher PAL activity (Bartsch et al., 2013). Aminomutases have been found in the biosynthetic pathways to antitumor drug compounds.
A number of tyrosine ammonia lyases have been cloned and functionally characterized: While PAL and TAL activities had been shown in plant extracts previously, Kyndt et al (Kyndt et al., 2002) identified and characterized the first TAL enzyme, originating from the purple non-sulfur bacterium Rhodobacter capsulatus, which uses pHCA as a chromophore in the light-sensing photoactive yellow protein (PYP). The actinomycete Saccharothrix espanaensis produce two related oligosaccharide antibiotics saccharomicin A and B, both containing a substructure derived from pHCA, which is formed by the sam8 gene of the antibiotic biosynthetic gene cluster (Berner et al., 2006; Strobel et al., 2012). EncP is a PAL playing a role in the biosynthetic pathway to enterocin in Streptomyces maritimus (Xiang; Moore, 2002), and recently, another TAL was identified in an actinomycete, namely bagA in Streptomyces sp. Tü 4128 (Zhu et al., 2012), and as a part of biosynthetic route to bagremycin A and B. stlA of Photorhabdus luminescens is also part of an antibiotic biosynthetic pathway, yet StlA has PAL activity (Williams et al., 2005). A number of the TALs have been purified and enzymatically characterized (Appert et al., 1994; Rosier et al., 1997; Kyndt et al., 2002; Christenson et al., 2003b; Williams et al., 2005; Berner et al., 2006; Schroeder et al., 2008; Bartsch; Bornscheuer, 2009).
TAL enzymatic activity has been described in patent literature and in particular the enzymes of the yeast genus Rhodotorula, the yeasts Phanerochaete chrysosporium and Trichosporon cutaneum, and the purple non-sulfur bacteria Rhodobacter sphaeroides and capsulatus. However, since these enzymes also show some specificity towards phenylalanine, they are not particularly useful in the production of p-coumaric acid and other hydroxycinnamic acids due to accompanying contamination by cinnamic acid as a result of the deamination of phenylalanine.
Accordingly, there is a need in the art for biological processes which allow the production of p-coumaric acid and other hydroxycinnamic acids at high yield and high purity. This need is solved by the present invention.